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The antibodies and dilutions for Immunohistochemical staining

Supplemental Figure 5 shows (A) treatment schedule of subcutaneous model, (B) therapeutic result of PK-9 subcutaneous tumor model mice (n= 5). 3 groups (PBS: non-treatment group, 11R-7_14 AAA: negative control peptide group, 11R-DB: treatment group). In the 11R-DB group, tumor growth was suppressed significantly (11R-DB vs 11R-7â,‹14AAA and PBS: *p

Supplemental Figure 2 shows, (A) analysis of expression of C16orf74 in PDAC and other cancer cell lines by qRT-PCR and (B) analysis of expression of endogenous C16orf74 protein in PDAC and NHDF by Western blot.

Supplemental Figure 4 shows the quantitative graph of AKt phosphorylation and mTOR phosphorylation. (A) AKt phosphorylation in PK-1, PK-9, PANC-1 and MIAPaCa-2 cells. (B) mTOR phosphorylation in PK-1, PK-9, PANC-1 and MIAPaCa-2 cells.

Supplemental Figure 1 shows, (A) analysis of expression of C16orf74 in C16orf74 stable and MOCK cell lines by qRT-PCR, (B) expression of C16orf74 in C16orf74 stable and MOCK by Western blotting, (C) expression of C16orf74 in C16orf74 stable clonal cell lines by Immunofluorescence. C16orf74 localizes under the cell membrane, and (D) expression of endogenous C16orf74 protein in PK-9 cells shown by Immunofluorescence. Endogenous C16orf74 localizes under the cell membrane.

Supplemental Figure 3 shows matrigel invasion assay of C16orf74 stable cell lines (Clone C16-A. C16-B. C16-C) with 11R-DB administration and no administration. In the administered group, the invasion was significantly suppressed. * P

The antibodies and dilutions for Immunofluorescence

The primary antibodies and dilutions for Western blot analysis

RT-PCR primers for construction of the expression vectors

Blood vessels deliver oxygen and nutrients to tissues, and vascular networks are spatially organized to meet the metabolic needs for maintaining homeostasis. In contrast, the vasculature of tumors is immature and leaky, resulting in insufficient delivery of nutrients and oxygen. Vasculogenic processes occur normally in adult tissues to repair “injured” blood vessels, leading us to hypothesize that bone marrow mononuclear cells (BMMNC) may be able to restore appropriate vessel function in the tumor vasculature. Culturing BMMNCs in endothelial growth medium resulted in the early outgrowth of spindle-shaped attached cells expressing CD11b/Flt1/Tie2/c-Kit/CXCR4 with proangiogenic activity. Intravenous administration of these cultured vascular proangiogenic cells (VPC) into nude mice bearing pancreatic cancer xenografts and Pdx1-Cre;LSL-KrasG12D;p53lox/+ genetically engineered mice that develop pancreatic ductal adenocarcinoma significantly reduced areas of hypoxia without enhancing tumor growth. The resulting vasculature structurally mimicked normal vessels with intensive pericyte coverage. Increases in vascularized areas within VPC-injected xenografts were visualized with an ultrasound diagnostic system during injection of a microbubble-based contrast agent (Sonazoid), indicating a functional “normalization” of the tumor vasculature. In addition, gene expression profiles in the VPC-transplanted xenografts revealed a marked reduction in major factors involved in drug resistance and “stemness” of cancer cells. Together, our findings identify a novel alternate approach to regulate abnormal tumor vessels, offering the potential to improve the delivery and efficacy of anticancer drugs to hypoxic tumors. Cancer Res; 70(15); 6283–92. ©2010 AACR.

Supplementary Figure 2 from Transplanting Normal Vascular Proangiogenic Cells to Tumor-Bearing Mice Triggers Vascular Remodeling and Reduces Hypoxia in Tumors

Supplementary Movie 1 from Transplanting Normal Vascular Proangiogenic Cells to Tumor-Bearing Mice Triggers Vascular Remodeling and Reduces Hypoxia in Tumors

Supplementary Table 1 from Transplanting Normal Vascular Proangiogenic Cells to Tumor-Bearing Mice Triggers Vascular Remodeling and Reduces Hypoxia in Tumors

Supplementary Figure 1 from Transplanting Normal Vascular Proangiogenic Cells to Tumor-Bearing Mice Triggers Vascular Remodeling and Reduces Hypoxia in Tumors

Supplementary Figure Legends 1-3 from Transplanting Normal Vascular Proangiogenic Cells to Tumor-Bearing Mice Triggers Vascular Remodeling and Reduces Hypoxia in Tumors

Supplementary Figure 3 from Transplanting Normal Vascular Proangiogenic Cells to Tumor-Bearing Mice Triggers Vascular Remodeling and Reduces Hypoxia in Tumors

Supplementary Table 2 from Transplanting Normal Vascular Proangiogenic Cells to Tumor-Bearing Mice Triggers Vascular Remodeling and Reduces Hypoxia in Tumors

Simple Summary Extrahepatic cholangiocarcinoma (EHCC) is an aggressive tumor. The five-year survival rate for patients who undergo surgical resection is only 20-40% due to recurrences. Therefore, elucidating the molecular mechanisms underlying invasion and metastasis in EHCC is crucial for developing adjuvant therapy. The epithelial-mesenchymal transition (EMT) contributes to the metastatic cascade in various tumors. C-C chemokine receptor 7 (CCR7) interacts with its ligand, chemokine (C-C motif) ligand 19 (CCL19), to promote EMT. The association between CCR7 expression and clinicopathological features and EMT status was examined via the immunohistochemical staining of tumor sections from 181 patients with perihilar cholangiocarcinoma. This association was then investigated in two EHCC cell lines. CCR7 mediates cell invasion and migration in EHCC by inducing EMT, which was abrogated by a CCR7 antagonist. CCR7 may be a potential target for adjuvant therapy in EHCC. The epithelial-mesenchymal transition (EMT) contributes to the metastatic cascade in various tumors. C-C chemokine receptor 7 (CCR7) interacts with its ligand, chemokine (C-C motif) ligand 19 (CCL19), to promote EMT. However, the association between EMT and CCR7 in extrahepatic cholangiocarcinoma (EHCC) remains unknown. This study aimed to elucidate the prognostic impact of CCR7 expression and its association with clinicopathological features and EMT in EHCC. The association between CCR7 expression and clinicopathological features and EMT status was examined via the immunohistochemical staining of tumor sections from 181 patients with perihilar cholangiocarcinoma. This association was then investigated in TFK-1 and EGI-1 EHCC cell lines. High-grade CCR7 expression was significantly associated with a large number of tumor buds, low E-cadherin expression, and poor overall survival. TFK-1 showed CCR7 expression, and Western blotting revealed E-cadherin downregulation and vimentin upregulation in response to CCL19 treatment. The wound healing and Transwell invasion assays revealed that the activation of CCR7 by CCL19 enhanced the migration and invasion of TFK-1 cells, which were abrogated by a CCR7 antagonist. These results suggest that a high CCR7 expression is associated with an adverse postoperative prognosis via EMT induction and that CCR7 may be a potential target for adjuvant therapy in EHCC.

Therapeutic efficacy of retroviral replicating vector (RRV)-mediated prodrug activator gene therapy has been demonstrated in a variety of tumor models, but clinical investigation of this approach has so far been restricted to glioma and gastrointestinal malignancies. In the present study, we evaluated replication kinetics, transduction efficiency, and therapeutic efficacy of RRV in experimental models of lung cancer. RRV delivering GFP as a reporter gene showed rapid viral replication in a panel of lung cancer cells in vitro, as well as robust intratumoral replication and high levels of tumor transduction in subcutaneous and orthotopic pleural dissemination models of lung cancer in vivo. Toca 511 (

Therapeutic efficacy of retroviral replicating vector (RRV)-mediated prodrug activator gene therapy has been demonstrated in a variety of tumor models, but clinical investigation of this approach has so far been restricted to glioma and gastrointestinal malignancies. In the present study, we evaluated replication kinetics, transduction efficiency, and therapeutic efficacy of RRV in experimental models of lung cancer. RRV delivering GFP as a reporter gene showed rapid viral replication in a panel of lung cancer cells in vitro, as well as robust intratumoral replication and high levels of tumor transduction in subcutaneous and orthotopic pleural dissemination models of lung cancer in vivo. Toca 511 (vocimagene amiretrorepvec), a clinical-stage RRV encoding optimized yeast cytosine deaminase (yCD) which converts the prodrug 5-fluorocytosine (5-FC) to the active drug 5-fluorouracil (5-FU), showed potent cytotoxicity in lung cancer cells upon exposure to 5-FC prodrug. In vivo, Toca 511 achieved significant tumor growth inhibition following 5-FC treatment in subcutaneous and orthotopic pleural dissemination models of lung cancer in both immunodeficient and immunocompetent hosts, resulting in significantly increased overall survival. This study demonstrates that RRV can serve as highly efficient vehicles for gene delivery to lung cancer, and indicates the translational potential of RRV-mediated prodrug activator gene therapy with Toca 511/5-FC as a novel therapeutic strategy for pulmonary malignancies.

Over the past 30 years, the therapeutic outcome for pancreatic ductal adenocarcinoma (PDAC) has remained stagnant due to the lack of effective treatments. We performed a genome-wide analysis to identify novel therapeutic targets for PDAC. Our analysis showed that Homo sapiens chromosome 16 open reading frame 74 (C16orf74) was upregulated in most patients with PDAC and associated with poor prognosis. Previously, we demonstrated that C16orf74 interacts with the catalytic subunit alpha of protein phosphatase 3 and plays an important role in PDAC invasion. However, the pathophysiologic function of C16orf74 is still unclear. In this study, through the analysis of C16orf74 interaction, we demonstrate a new strategy to inhibit the growth and invasion of PDAC. C16orf74 exists in the homodimer form under the cell membrane and binds integrin αVβ3 and is also involved in invasion by activating Rho family (Rac1) and MMP2. Considering that this dimeric form was found to be involved in the function of C16orf74, we designed an 11R-DB (dimer block) cell-permeable dominant-negative peptide that inhibits the dimer form of C16orf74. 11R-DB suppressed invasion and proliferation of PDAC cell lines by inhibiting phosphorylation of Akt and mTOR and also by inactivation of MMP2. 11R-DB also showed antitumor effects in an orthotopic xenograft model and peritoneal metastasis model. Thus, this study demonstrates that dimerized C16orf74, present in the cell membrane, is involved in pancreatic cancer invasion and proliferation. In addition, the C16orf74 dimer block cell-permeable peptide (11R-DB) has a potent therapeutic effect on PDAC in vitro and in vivo.

Simple Summary Pancreatic ductal adenocarcinoma is one of the most lethal malignancies, and there are vast unmet medical needs. In this study, we hypothesized that chimeric antigen receptor engineered T cell (CAR-T) targeting carcinoembryonic antigen (CEA) would be effective in the treatment of pancreatic ductal adenocarcinoma. In vivo experiments in a more clinically similar environment were considered necessary; we examined the antitumor effects of adoptive anti-CEA-CAR-T, using orthotopic xenograft mouse models of pancreatic ductal adenocarcinoma. As result, the therapeutic effect of anti-CEA-CAR-T therapy was related to the CEA expression level. Furthermore, the retrospective analysis of pathological findings from pancreatic ductal adenocarcinoma patients showed a correlation between the intensity of CEA immunostaining and tumor heterogeneity. These findings show that anti-CEA-CAR-T therapy can be useful for pancreatic ductal adenocarcinoma; furthermore, the pathological findings of CEA can be clinically used as biomarkers to select cases for anti-CEA-CAR-T therapy. Chimeric antigen receptor engineered T cell (CAR-T) therapy has high therapeutic efficacy against blood cancers, but it has not shown satisfactory results in solid tumors. Therefore, we examined the therapeutic effect of CAR-T therapy targeting carcinoembryonic antigen (CEA) in pancreatic adenocarcinoma (PDAC). CEA expression levels on the cell membranes of various PDAC cell lines were evaluated using flow cytometry and the cells were divided into high, medium, and low expression groups. The relationship between CEA expression level and the antitumor effect of anti-CEA-CAR-T was evaluated using a functional assay for various PDAC cell lines; a significant correlation was observed between CEA expression level and the antitumor effect. We created orthotopic PDAC xenograft mouse models and injected with anti-CEA-CAR-T; only the cell line with high CEA expression exhibited a significant therapeutic effect. Thus, the therapeutic effect of CAR-T therapy was related to the target antigen expression level, and the further retrospective analysis of pathological findings from PDAC patients showed a correlation between the intensity of CEA immunostaining and tumor heterogeneity. Therefore, CEA expression levels in biopsies or surgical specimens can be clinically used as biomarkers to select PDAC patients for anti-CAR-T therapy.

The efficacy of preoperative neoadjuvant chemoradiotherapy (NAC) in cases of pancreatic cancer with extremely poor prognoses has been reported. In this study, we aimed to identify novel biomarkers that reflect prognoses following chemoradiotherapy using tertiary lymphoid organs (TLO) expressed in the tumor microenvironment. Resected tumor specimens were obtained from 140 pancreatic cancer patients. We retrospectively investigated the clinical relevance of TLO by categorizing patients into those who underwent upfront surgery (surgery first [SF]) and those who received NAC. The immunological elements within TLO were analyzed by immunohistochemistry (IHC). In the IHC analysis, the proportions of CD8+ T lymphocytes, PNAd+ high endothelial venules, CD163+ macrophages and Ki‐67+ cells within the TLO were higher in the NAC group than in the SF group. In contrast, the proportion of programmed cell death‐1+ immunosuppressive lymphocytes within TLO was lower in the NAC group than in the SF group. The NAC group demonstrated favorable prognoses compared with the SF group. In the multivariate analysis, the TLO/tumor ratio was determined as an independent predictive prognostic factor. In conclusion, the administration of preoperative chemoradiotherapy may influence the immunological elements in the tumor microenvironment and result in favorable prognoses in pancreatic ductal adenocarcinoma patients.

Cancer stem cells (CSCs) are specific targets for therapeutic applications, but the rarity of CSCs within tumors makes the isolation of CSCs difficult. To overcome these problems, we generated CSCs in vitro using established reprogramming techniques. We transduced four previously established reprogramming factors, Oct3/4, Sox2, Klf4, and L-myc, into the colon cancer cell lines LoVo and OUMS-23, and investigated the biological characteristics of these lines. Tra-1-60+ cells were obtained from reprogrammed induced pluripotent stem (iPS) cell-like colonies and showed CSC properties, including colony formation, maintenance of colonies by repeated passages, and feeder cell dependency, as well as increased expressions of CSC markers such as CD133 and ALDH1. The CSC-like cells showed increased chemoresistance to 5-fluorouracil and elevated tumorigenicity upon transplantation into kidneys of immune-deficient mice. These tumors shifted to a poorly differentiated stage with many atypical cells, cytoplasmic mucin, and focal papillary components, with demonstrated dedifferentiation. The principal component analysis from DNA microarrays showed that though both cell lines moved to iPS cells after reprogramming, they were not completely identical to iPS cells. Significantly elevated gene expression of Decorin and CD90 was observed in CSC-like cells. Together, these results show that reprogramming of cancer cells produced not pluripotent stem cells but CSC-like cells, and these findings will provide biological information about genuine CSCs and help establish new CSC-targeted therapies.

A new, simple and inexpensive method is proposed to determine the rate of water filtration in the swash zone of lakeshore beaches. Rates of water filtration on a sandy beach were determined by measuring the residue of a tracer injected into several cylinders buried vertically in the sandy layer at various locations from the shoreline to the tip of the swash zone. This methodology enabled the separate estimation of mean infiltration and exfiltration rates. The results obtained for seashore beaches were in accordance with previous data obtained by more conventional methods. The filtration rate results for lakeshore sandy beaches were almost equivalent to seashore sandy beaches, despite a shorter swash length. This paper indicates that this is caused by a larger grain size and shorter wave period. This method presents an easy approach to determine rates of water filtration in sandy beaches and is expected to further promote studies in this field.

It has been clear that the clinical responses by applying immune checkpoint inhibitor alone are limited. To better improve this limited clinical response, combinational therapy has been focused. We recently reported the association between EMT related factors and PD-L1 expression in the extrahepatic hilar cholangiocarcinoma, and its role as a surrogate biomarker for patient prognosis. We here report clinical relevance of combinational therapy of HDAC inhibitors and anti-PD-L1 antibody as an immune checkpoint inhibitors.

Hepcidin is a main regulator of iron metabolism, of which abnormal expression affects intestinal absorption and reticuloendothelial sequestration of iron by interacting with ferroportin. It is also noted that abnormal iron accumulation is one of the key factors to facilitate promotion and progression of cancer including hepatoma. In this study, we firstly revealed that a new alternative HAMP transcript was found in hepatoma-derived cell line HLF, which was identical to the wild-type preprohepcidin sequence except lacking of an internal 60 bases. In addition to HLF, most of hepatoma-derived cell lines have significant copy numbers of variant-type hepcidin mRNA by a copy-based-digital PCR. Furthermore, the copy number of hepcidin mRNA variant was significantly higher in serum exosomes of hepatocellular carcinoma patients. The quantification of exosomal hepcidin mRNA variant may serve as a potential new biomarker for HCC diagnosis.

We established mutated and non-mutated induced pluripotent stem cell (iPSC) clones from a patient with PTPN11 (c.226G>A)-mutated juvenile myelomonocytic leukaemia (JMML). Both types of iPSCs fulfilled the quality criteria. Mutated iPSC colonies generated significantly more CD34+ and CD34+ CD45+ cells compared to non-mutated iPSC colonies in a culture coated with irradiated AGM-S3 cells to which four growth factors were added sequentially or simultaneously. The haematopoietic differentiation potential of non-mutated JMML iPSC colonies was similar to or lower than that of iPSC colonies from a healthy individual. The PTPN11 mutation coexisted with the OSBP2 c.389C>T mutation. Zinc-finger nuclease-mediated homologous recombination revealed that correction of PTPN11 mutation in iPSCs with PTPN11 and OSBP2 mutations resulted in reduced CD34+ cell generation to a level similar to that obtained with JMML iPSC colonies with the wild-type of both genes, and interestingly, to that obtained with normal iPSC colonies. Transduction of the PTPN11 mutation into JMML iPSCs with the wild-type of both genes increased CD34+ cell production to a level comparable to that obtained with JMML iPSC colonies harbouring the two genetic mutations. Thus, PTPN11 mutation may be the most essential abnormality to confer an aberrant haematopoietic differentiation potential in this disorder.

Hepcidin is a main regulator of iron metabolism, of which abnormal expression affects intestinal absorption and reticuloendothelial sequestration of iron by interacting with ferroportin. It is also noted that abnormal iron accumulation is one of the key factors to facilitate promotion and progression of cancer including hepatoma. In this study, we firstly revealed that a new alternative HAMP transcript was found in hepatoma-derived cell line HLF, which was identical to the wild-type preprohepcidin sequence except lacking of an internal 60 bases. In addition to HLF, most of hepatoma-derived cell lines have significant copy numbers of variant-type hepcidin mRNA by a copy-based-digital PCR. Furthermore, the copy number of hepcidin mRNA variant was significantly higher in serum exosomes of hepatocellular carcinoma patients. The quantification of exosomal hepcidin mRNA variant may serve as a potential new biomarker for HCC diagnosis.

The final publication is available at Springer via http://dx.doi.org/10.1007/s12185-016-2054-7, Iron overload remains a concern in myelodysplastic syndrome (MDS) patients. Iron chelation therapy (ICT) thus plays an integral role in the management of these patients. Moreover, ICT has been shown to prolong leukemia-free survival in MDS patients; however, the mechanisms responsible for this effect are unclear. Iron is a key molecule for regulating cytosolic aconitase 1 (ACO1). Additionally, the mutation of isocitrate dehydrogenase (IDH), the enzyme downstream of ACO1 in the TCA cycle, is associated with epigenetic abnormalities secondary to 2-hydroxyglutarate (2-HG) and DNA methylation. However, epigenetic abnormalities observed in many MDS patients occur without IDH mutation. We hypothesized that iron itself activates the ACO1-IDH pathway, which may increase 2-HG and DNA methylation, and eventually contribute to leukemogenesis without IDH mutation. Using whole RNA sequencing of bone marrow cells in iron-overloaded mice, we observed that the enzymes, phosphoglucomutase 1, glycogen debranching enzyme, and isocitrate dehydrogenase 1 (Idh1), which are involved in glycogen and glucose metabolism, were increased. Digital PCR further showed that Idh1 and Aco1, enzymes involved in the TCA cycle, were also elevated. Additionally, enzymatic activities of TCA cycle and methylated DNA were increased. Iron chelation reversed these phenomena. In conclusion, iron activation of glucose metabolism causes an increase of 2-HG and DNA methylation.

The cardiac portion of the stomach is situated deeply in the upper abdomen and relatively well hidden. The topographic space around it is very narrow and complicated. These topographical features are produced by the liver, especially segments I, II, and III. An understanding of the relationships among compacted organs facilitates operative access by surgeons.

Estradiol (E2) is one of the sex steroid hormones, whose various well-known functions include control of the female reproductive system, causing breast cancers, and inhibiting arterial sclerosis. In this study to investigate how E2 affected cell differentiation of induced pluripotent stem cells (iPSCs), we used different methods to culture hiPSCs, the undifferentiated state, longterm culture without passages (natural differentiation), embryoid bodies (EBs), and accelerated differentiation by activin, focusing on marker gene expression, including Brachyury, hepatocyte nuclear factor (HNF) 3β, estrogen receptor α, and other markers. When iPSCs began to differentiate by responding to E2, Brachyury expression decreased regardless of the amount of E2, cell line, or protocol. Other markers were up or down-regulated, but did not show any tendency except in the early stage of EBs. The definite timing that E2 had the strongest effect on the aspect of differentiation was the EB stage analogous to gastrulation, in which E2 repressed Brachyury expression and induced HNF3β at its restricted amount. These results suggest that the first target of E2 is the gastrulation stage.

N-Benzyl-substituted phenethylamines (NBOMes) have emerged as novel hallucinogenic designer drugs with potent serotonin-receptor activation. We present the first scientific case report of fatal intoxication with 2-(4-bromo-2,5-dimethoxyphenyl)-N-(2-methoxybenzyl)ethanamine (25B-NBOMe). The plasma concentration of 25B-NBOMe upon admission was low, 3.15 ng/ml, but it exceeded concentrations reported previously in NBOMe intoxications cases. Our case documents complete clinical and pathophysiological findings that developed shortly after drug ingestion and highlights the danger of 25B-NBOMe use at small doses, based on autopsy and toxicological analyses. The patient presented symptoms consistent with serotonin syndrome.

The fenestrations of liver sinusoidal endothelial cells (LSECs) play important roles in the exchange of macromolecules, solutes, and fluid between blood and surrounding liver tissues in response to hepatotoxic drugs, toxins, and oxidative stress. As excess iron is a hepatotoxin, LSECs may be affected by excess iron. In this study, we found a novel link between LSEC defenestration and hepatic nerve growth factor (NGF) in iron-overloaded mice. By Western blotting, NGF was highly expressed, whereas VEGF and HGF were not, and hepatic NGF mRNA levels were increased according to digital PCR. Immunohistochemically, NGF staining was localized in hepatocytes, while TrkA, an NGF receptor, was localized in LSECs. Scanning electron microscopy revealed LSEC defenestration in mice overloaded with iron as well as mice treated with recombinant NGF. Treatment with conditioned medium from iron-overloaded primary hepatocytes reduced primary LSEC fenestrations, while treatment with an anti-NGF neutralizing antibody or TrkA inhibitor, K252a, reversed this effect. However, iron-loaded medium itself did not reduce fenestration. In conclusion, iron accumulation induces NGF expression in hepatocytes, which in turn leads to LSEC defenestration via TrkA. This novel link between iron and NGF may aid our understanding of the development of chronic liver disease.

To demonstrate that intra-organ splenosis can engraft and develop after being distributed through the vasculature, tiny fragments of splenic tissues were injected into the inferior vena cava or the portal vein to induce intrapulmonary and intrahepatic splenosis in rats. After 1 month, splenic autograft structures in the lung and liver were assessed for structure by histology, for immunologic compartments by immunohistochemistry, for phagocytic function by carbon uptake and for vascular formation by Microfil (a silicon rubber compound) injection. Intrapulmonary and intrahepatic splenoses were indeed able to spread through the vasculature. The intrapulmonary splenic autografts were trapped and spread out in the interstitium, without forming a capsule. White pulp was markedly developed, showing lymphocyte aggregations that consisted in B cells surrounding the dilated vessel. Splenic sinuses were not definitively observed. Although macrophages were detected by immunohistochemistry, they showed no indication of having phagocytized carbon particles from the vessels, implying a closed circulation. In contrast, intrahepatic splenic autografts formed well-developed capsules, trabeculae and red pulp with splenic sinuses. Macrophages detected by immunohistochemistry were observed capturing carbon particles, which clearly revealed an open system circulation, as seen in normal rat spleen. The development of white pulp was poor and lymphocytes consisting in B cells aggregated in the peripheral margins. These results demonstrate that intra-organ splenosis can spread through the vasculature and that the morphologic and immunologic structures formed in these regenerated autografts are influenced by the organ vasculature and extracellular matrix wherein the tissue fragments settled.

author, Iron is an essential metal in the body, but its excessive accumulation causes damage in various organs through free radical production. Iron homeostasis is therefore tightly regulated. However, when iron balance collapses, such as in prolonged transfusion, transferrin (Tf) is fully saturated and non-Tf-bound iron (NTBI) appears in the serum. Monitoring serum NTBI levels is therefore crucial in assessment of the clinical status of patients with iron overload, since NTBI is associated with cellular and organ damage. Several methods for NTBI determination have been reported, but these are extremely complicated and very few laboratories can quantify NTBI at present. In this study, we established a novel assay system utilizing automated analyzers that are widely used in clinical laboratories for diagnostic testing. In this assay, NTBI is chelated by nitrilotriacetic acid (NTA), after which the iron is reduced and transferred to nitroso-PSAP, a chromogen. The assay shows excellent linearity, reproducibility, and compatibility with HPLC, one of the most reliable conventional methods for NTBI quantification. Our novel method for NTBI measurement is high-throughput and may be a useful and powerful tool in study of the physiological and clinical importance of NTBI.

The gastric cardia—the small area around the cardiac orifice including the abdominal esophagus—is an important target area for abdominal and thoracic surgeries, especially for laparoscopic procedures. In this study of 28 cadavers, a peritoneal earlobe-like appendage near the angle of His was identified as a useful indicator of the lateral margin of the abdominal esophagus, which is otherwise obscure because the peritoneum continues to the diaphragm without definite demarcation of this margin. This structure, which appears equivalent to the epiploic appendages, was commonly found to be present (in 22/28, 78.6 % of the 28 cadavers) and was 4–21 mm × 6–40 mm × 1–4 mm in size, triangular, round, or leaf-like in shape, contained fat, and was on an imaginary line along which the lesser omentum adheres to the lesser curvature and continues to the diaphragm (18/22, 81.8 %). This indicator is associated with the lesser omentum and is part of the gastrophrenic ligament, and could serve as a useful indicator of the margin of the gastric cardia, thus aiding surgeons performing laparoscopic surgery in this region.

IntroductionReducing uncertainty about information on injury severity or number of patients is an important concern in managing equipment and rescue personnel in a disaster setting. A simplified disaster model was designed using Shannon’s Information Theory to study the uncertainty of information in a triage scenario.HypothesisA disaster triage scene with a specific number of injured patients represents a source of information regarding the extent of patients’ disability. It is possible to quantify uncertainty of information regarding patients’ incapacity as entropy if the information source and information arising from the source in Information Theory can be adapted to the disaster situation and the information on patients’ incapacity that arises.MethodsFive different scenarios of a fire disaster in a hospital were modeled. Information on patients’ extent of impairment was converted to numerical values in relation to available equipment and the number of rescue personnel. Victims were 10 hospitalized patients with conditions of unknown severity. Triage criteria were created arbitrarily and consisted of four categories from Level 1 (able to walk) to Level 4 (cardiac arrest). The five situations were as follows: (1) Case 1: no triage officer; (2) Case 2: one triage officer; (3) Case 3: one triage officer and a message that six patients could walk; (4) Case 4: one triage officer and a message that all patients could obey commands; and (5) Case 5: one triage officer and a message that all patients could walk. Entropy in all cases and the amount of information newly given in Cases 2 through 5 were calculated.ResultsEntropies in Cases 1 through 5 were 5.49, 2.00, 1.60, 1.00, and 0.00 bits/symbol, respectively. These values depict the uncertainty of probability of the triage categories arising in each situation. The amount of information for the triage was calculated as 3.49 bits (ie, 5.49 minus 2.00). In the same manner, the amount of information for the messages in Cases 3 through 5 was calculated as 0.4, 1.0, and 2.0 bits, respectively. These amounts of information indicate a reduction in uncertainty regarding the probability of the triage levels arising.ConclusionIt was possible to quantify uncertainty of information about the extent of disability in patients at a triage location and to evaluate reduction of the uncertainty by using entropy based on Shannon’s Information Theory.AjimiY, SasakiM, UchidaY, GakumazawaM, SasakiK, FujitaT, SakamotoT. Quantitative evaluation for uncertainty of information about patients’ injury severity in a hospital disaster: a simulation study using Shannon’s Information Theory. Prehosp Disaster Med. 2015;30(4):1-4.

AIM: To facilitate close contacts between transplanted cardiomyocytes and host skeletal muscle using cell fusion mediated by hemagglutinating virus of Japan envelope (HVJ-E) and tissue maceration. METHODS: Cardiomyocytes (1.5 × 106) from fetal rats were first cultured. After proliferation, some cells were used for fusion with adult muscle fibers using HVJ-E. Other cells were used to create cardiomyocyte sheets (area: about 3.5 cm2 including 2.1 × 106 cells), which were then treated with Nile blue, separated, and transplanted between the latissimus dorsi and intercostal muscles of adult rats with four combinations of HVJ-E and/or NaOH maceration: G1: HVJ-E(+), NaOH(+), Cardiomyocytes(+); G2: HVJ-E(-), NaOH(+), Cardiomyocytes(+); G3: HVJ-E(+), NaOH(-), Cardiomyocytes(+); G4: HVJ-E(-), NaOH(-), Cardiomyocytes(-). At 1 and 2 wk after transplantation, the four groups were compared by detection of beating domains, motion images using moving target analysis software, action potentials, gene expression of MLC-2v and Mesp1 by reverse transcription-polymerase chain reaction, hematoxylin-eosin staining, and immunostaining for cardiac troponin and skeletal myosin. RESULTS: In vitro cardiomyocytes were fused with skeletal muscle fibers using HVJ-E. Cardiomyocyte sheets remained in the primary transplanted sites for 2 wk. Although beating domains were detected in G1, G2, and G3 rats, G1 rats prevailed in the number, size, motion image amplitudes, and action potential compared with G2 and G3 rats. Close contacts were only found in G1 rats. At 1 wk after transplantation, the cardiomyocyte sheets showed adhesion at various points to the myoblast layer in the latissimus dorsi muscle. At 2 wk after transplantation, close contacts were seen over a broad area. Part of the skeletal muscle sarcoplasma seemed to project into the myocardiocyte plasma and some nuclei appeared to share both sarcoplasmas. CONCLUSION: The present results show that close contacts were acquired and facilitated the beating function, thereby providing a new cellular transplantation method using HVJ-E and NaOH maceration.

In this study, image processing was applied to photographs of the road surface taken from a traveling vehicle using a simple and inexpensive photographing unit, and method for automatic detection of pavement crack was developed. In comparison of the automatic detection and visual detection, the concordance rate of the crack detection by both methods became more than 80%. Furthermore, the authors calculated crack progress level (small, middle, large) using the automatically detecting method and compared it with the result of road surface measuring vehicle. It was revealed that the concordance rate of the crack detection by the both methods became more than 90%. 舗装路面のひび割れ調査には路面性状測定車が広く用いられているが，これには多額の費用が必要となるため，市町村レベルでは調査を実施することが難しい．本研究では，走行車両から市販のデジタルカメラで撮影した路面画像にアフィン変換や動的2値化処理などの画像解析を適用して自動的にひび割れを検出する手法を開発した．自動検出によって得られた結果と目視によってひび割れを検出した結果の一致率は80%以上であった．さらに，本手法を用いて総点検実施要領【舗装編】に示されたひび割れレベル（小・中・大）を求め，これと路面性状測定車から得られた値と比べたところ，80%以上の一致率が得られた．